Antibody-directed enzyme prodrug therapy (ADEPT) is a promising anti- cancer chemotherapeutic strategy in which enzymes are concentrated at the tumor site when administered as conjugates of tumor-specific antibodies. After unbound conjugate has cleared from the circulation, prodrugs may be administered which are non-toxic until activated by the tumor-bound enzyme. Beta-lactamase has been used with cephalosporin prodrugs of aniline mustards and anthracyclines to achieve promising anti-tumor effects in animals. The efficacy of ADEPT is limited, however, by the need for unbound conjugate to clear the circulation before the prodrug can be administered, by which time so much of the conjugate has been lost from the tumor that efficacy is compromised. To address this problem we have developed an interaction-dependent fragment complementation system for beta-lactamase. From a library of all possible combinations beta-lactamase fragments, we have isolated fragment pairs which complement to form the active enzyme only when the fragments are fused to proteins which interact with one another directly or via a third molecule. When fused to antibody fragments which recognize non-overlapping epitopes on tumor markers, these fragments should be able to reconstitute sufficient beta- lactamase activity on the tumor cell surface to produce unprecedented levels of tumor-localized cytotoxicity from beta-lactam prodrugs. PROPOSED COMMERCIAL APPLICATIONS: The primary goal of this work is to develop a new and effective strategy for the chemotherapeutic treatment of human cancer.